Project description:Purpose: Nxf1 is thought to be an essential nuclear exporter of messenger RNA (mRNA) in eukaryotic cells. Whether perturbations in the Nxf1 pathway affect mammalian physiology is not known. The aim of this study is to determine the impact of a Nxf1 mutation in the representation of mRNA transcripts in the cytoplasm of naïve CD8 T cells. Methods: For Naïve CD8 T cell isolation, splenocyte suspensions were submitted to red blood cell lysis, stained first with biotinylated CD8 antibodies and then labelled with anti-Biotin microbeads. CD8+ T cells were magnetically enriched by positive selection on MACS separation columns, stained with fluorescently-labelled antibodies before being FACS-sorted as CD8+ CD44low CD62L+. Sorted Naïve T cells were fractionated with Norgen cytoplasmic and nuclear fractionation RNA purification kit. Only cytoplasmic fractions were used for subsequent RNAseq.
Project description:In vitro and in vivo data from our lab show that TReg cells exhibit a right-shifted vedolizumab binding profile compared with TEff cells. Consistently, in a certain concentration range, the residual adhesion, transmigration and homing of TReg cells was higher than that of TEff cells. To better understand why vedolizumab binding was different between TReg and TEff in a certain concentration range, we performed single cell RNA sequencing of Memory CD4 T cells expressing α4 and β7 integrin and compared cells binding to vedolizumab or not binding to vedolizumab (previously purified using FACS with fluorescently labelled vedolizumab).
Project description:To investigate the mechanisms of cancer cell migration during perineural invasion in pancreatic cancer, we injected fluorescently labelled MiaPaCA-2 cells in the sciatic nerves and collected the leader and lagger cells.
Project description:In order to understand gene function, cells mutated for a gene need to be compared with normal cells. In most biomedical studies this comparative analysis is carried out with cells present in different animals and therefore not experiencing the same microenvironment or epigenetic changes. Here we present a large set of new genetic tools and mouse lines, that enable the Flp recombinase-dependent ratiometric induction and single cell clonal tracking of multiple fluorescently labelled normal and Cre-mutant cells, from distinct or the same progenitor cells. The labelled cells can be profiled in situ by multispectral imaging, or by FACS and scRNA-seq. With these new tools, normal and mutant cells can be ratiometrically induced and multispectrally barcoded within the same temporal window and tissue microenvironment. This enables a better understanding of how induced genetic mutations affect the biology of single cells with higher accuracy and reliability during tissue development, homeostasis, or disease.
Project description:We established simple synthetic microbial communities in a microcosm model system to determine the mechanisms that underlay cross-feeding in microbial methane-consuming communities. Co-occurring strains from Lake Washington sediment were used that are involved in methane consumption, a methanotroph and two non-methanotrophic methylotrophs.
Project description:Purpose: Investigate cellular heterogeneity in a fresh human ovarian cancer tissue sample Methods: Enzymatic digestion of fresh tissue sample collected from the operating room to produce single cell suspension. Cells were labelled with fluorescent antibodies to CD3, CD14, CD19, CD20, CD56 and FACS sorted to remove immune cells. The negative population was used for sequencing. Single cells were processed using the Fluidigm C1 Chip to generate barcoded cDNA for each cell. Amplifed cDNA was sequenced using an Illumina HiSeq 2500 machine. Results: Single cell RNA sequence data was obtained for 92 cells and a "bulk" sample of 1000 cells. 26 cells were removed from analysis due to quality control standards. The remaining 66 cells and the bulk sample were analyzed. Conclusion: Single cell RNA sequence analysis reveals heterogeneity in gene expression in cells harvested from a high grade ovarian serous cancer
Project description:Using yeast strains based on the protease-deficient strain BJ5459 (= wild type), the effects of the spt10-null and spt10-C388S mutations on global gene expression were assessed and compared. BJ5459 (wild type) and BJ-SPT10(C388S)-HA cells were grown in synthetic complete medium to A600 = 0.6. RNA was prepared using an RNeasy kit (Qiagen). Biotinylated cRNA was prepared from three independently prepared sample pairs and hybridised to Affymetrix S98 arrays and detected using fluorescently labelled streptavidin. The wild type and the spt10-C388S average signals each represent the average of the signals from three arrays. The P-value is an indicator of the statistical significance of the difference between the average signals for wild type and the C388S mutant. The lower the value, the greater the significance. The gene descriptor is provided by Affymetrix. Keywords: mutant yeast strain comparison
Project description:The interaction of lung epithelial and lung mesenchymal cells (fibroblasts) was investigated in a novel co-culture model of human pulmonary fibrosis. Remarkably, co-culturing both cell types induced cell-type-specific responses, including fibroblast-to-myofibroblast differentiation and epithelial-to-mesenchymal transition (EMT), which were fully dependent on direct epithelial / fibroblast contact. Primary normal human lung fibroblasts (NHLF) and normal human bronchiolar epithelial cells (NHBE) were fluorescently labelled prior to seeding, then grown either as mono or co-cultures and collected in triplicates for mRNA-seq at the start (T=0h) and after 3h and 18h. Each harvested cell culture was FACS sorted into individual NHLF and NHBE cell populations, which were then then lysed and sequenced.
